Non-small-cell lung cancer (NSCLC), accounting for about 85% of all lung cancers, is the leading cause of cancer deaths in the United States (Jemal et al., 2008) and worldwide. Despite advances in early detection and surgical resection, NSCLC often has a high recurrence.
KRAS is an oncogene located on Chromosome 12 (Ch. 12), with a cytogenic location of Ch. 12p12.1. KRAS encodes a protein called K-Ras that is involved in regulating cell division. The K-Ras protein has guanosinenucleotide-binding activity and intrinsic guanosine triphosphatase (GTPase) activity. K-Ras is downstream of epidermal growth factor receptor (EGFR), which signals through the PI3K/AKT/mTOR and STAT pathways involved in cell survival, and the RAS/RAF/MEK/MAPK pathway involved in cell proliferation.
The genetic code is a set of rules by which a gene is translated into a functional protein. Each gene includes a specific sequence of nucleotides encoded in a DNA (or sometimes RNA) strand. The four nucleotides are named for the heterocyclic base associated with them: adenine (“A”), cytosine (“C”), guanine (“G”), and thymine (“T”). The nucleotides polymerize to form a single strand of DNA, then two single strands interact by hydrogen bonding between complementary nucleotide, A being complementary with T and C being complementary with G, to form base pairs with results in the formation of a DNA double helix. RNA is similar to DNA except that the base thymine is replaced with uracil (“U”) and does not form double strands.
A gene can contain coding and/or non-coding DNA sequences that are transcribed into RNA. RNA sequences that are transcribed by coding sequences of a gene are known as messenger RNA (mRNA). mRNA sequences in turn encode for a particular proteins by the process of translation. Proteins produced from genes then perform a specific biochemical or structural function. A correspondence between nucleotides, the basic building blocks of genetic material, and amino acids, the basic building blocks of proteins, must be established for genes to be successfully translated into functional proteins. Sets of three nucleotides, known as codons, each correspond to a specific amino acid or to a signal; three codons are known as “stop codons” wherein, instead of specifying a new amino acid, alert the translation machinery that the end of the gene has been reached. There are 64 possible codons (four possible nucleotides at each or three positions) and only 20 standard amino acids. Thus, the code is redundant and multiple codons can specify the same amino acid.
RNA sequences that are transcribed by non-coding sequences of a gene are known as non-coding RNA (ncRNA), and are not translated into proteins. There are several types of ncRNA that are involved in various cellular functions. For example, transfer RNA (tRNA) and ribosomal RNA (rRNA) are involved in protein assembly, small nuclear RNA (snRNA) and ribozymes are involved in post-transcriptional processing and splicing of mRNA, and MicroRNAs (miRNA), Piwi-interacting RNA (piRNA) and small interfering RNA (siRNA) are involved in gene regulation by acting via the RNA interference (RNAi) system. The RNAi system involves miRNA, siRNA, piRNA or other RNA molecules that are complementary to a target DNA and/or RNA sequence, and regulates gene expression in several ways. For example, an miRNA, siRNA or piRNA may bind to and effect or accelerate the degradation of a target mRNA, or may bind to a target DNA or RNA sequence to block or enhance transcription or translation, respectively.
A variance, also known as a polymorphism or mutation, in the genetic code for any coding or non-coding gene sequence may result in the production of a gene product, usually a protein or an RNA molecule, with altered biochemical activity or with no activity at all, or may influence the function of that gene or locus. This can result from as little change as an addition, deletion, or substitution of a single nucleotide in the DNA comprising a particular gene that is sometimes referred to as a single nucleotide polymorphism (SNP).
Somatic mutations in the KRAS gene are involved in the development of many types of cancer, including NSCLC. When mutated in codon 12, 13 or 61, the KRAS genes encode a constitutively active K-Ras protein that continuously activate transducer signals by linking tyrosine kinases to downstream serine and threonine kinases. Activating point mutations have been found in various malignancies, including NSCLC. In advanced NSCLC, tumors that harbor KRAS point mutations have been correlated with progression of the disease, but not with survival (Massarelli et al., 2007). While the EGFR tyrosine kinase inhibitors, gefitinib and erlotinib can be beneficial for some NSCLC patients, the presence of KRAS mutations predicts primary resistance to these drugs (Massarelli et al., 2007; Zhu et al., 2008; Herbst et al., 2008).
In addition, variations in gene dosage, the number of copies of a gene that are present in a cell, can be clinically significant indicators of disease states. Such variations arise from errors in DNA replication and can occur in germ line cells (leading to congenital defects and even embryonic demise), or in somatic cells. These replication anomalies can cause deletion or duplication of parts of genes, full-length genes and their surrounding regulatory regions, megabase-long portions of chromosomes, or entire chromosomes.
Chromosomal abnormalities affect gene dosage on a larger scale and can affect either the number or structure of chromosomes. Conditions wherein cells, tissues, or individuals have one or more whole chromosomes or segments of chromosomes either absent, or in addition to the normal euploid complement of chromosomes can be referred to as aneuploidy.
Chromosomal aberrations in somatic cells, such as large deletions, insertions or amplifications that are the result of acquired mutations such as loss of heterozygosity (LOH) or gene duplication are associated with many diseases, including many types of cancer. Because somatic KRAS gene mutations have been associated with the development of many types of cancer, including NSCLC, chromosomal aberrations of the KRAS gene are also likely to be clinically relevant in cancer research. Detection of such chromosomal aberrations may have therapeutic, diagnostic or prognostic implications in cancer patients.
Methods for the detection of point mutations and small deletions or insertions in genomic DNA have been well established, however, detection of larger genomic deletions or other aberrations is more complicated. Chromosomal aberrations can be detected in cancer through chromosomal banding (Mertens et al., 1997; Database of Aberrations in cancer, found at http://cgap.nci.nih.gov/Chromosomes/Mitelman), fluorescent in situ hybridization (FISH) (Schrock et al., 1996; Fauth and Speicher, 2001; Speicher and Ward, 1996), and comparative genomic hybridization (CGH) (Kallioniemi et al., 1994; Pinkel et al., 1998). However, early detection of deletions and amplifications are difficult, largely because 1) there is a low frequency of aberrations in early stages of cancer development, 2) tumors often have a multiploid cancer genome, and 3) early stage cancer tissue specimens often have low proportions of tumor cells. Therefore, there is a need to develop more accurate and reliable methods to detect chromosomal deletions and aberrations in early stages of cancer, which may be used in the detection and discovery of predictive biomarkers in cancer.